Summary of Work: The aim of this project is to determine the causes of infertility in male mice with a targeted mutation of the estrogen receptor alpha gene. These studies will also help to define the role of estrogen in male gamete formation and function, and to identify estrogen-regulated genes in the testis and epididymis. The estrogen receptor is a ligand-dependent transcription factor that is essential for regulating female reproductive function, but its role in the male is poorly understood. Our studies found that in adult male estrogen receptor alpha knock-out (ERKO) mice, serum LH and FSH levels were normal, testosterone levels were elevated, and sperm numbers, the percent of sperm that were motile, testis weights, and mating frequencies were reduced. The lumen of the seminiferous tubules were dilated by 20 days of age and began degenerating by 70-90 days of age. The dilation of the tubules may be due to reduced fluid reabsorption in the efferent ducts connecting the testis to the epididymis. Approximately 85% of the fluid leaving the testis is reabsorbed in the efferent ducts by a sodium pump mechanism and the epithelium of these tubules contains higher levels of estrogen receptor than other male reproductive tract tissues. The number and motility of sperm in the epididymis of ERKO mice declined from 8 to 16 weeks of age and the sperm were unable to fertilize eggs in vitro, indicating that infertility was due to altered sperm function, in addition to reduced sperm numbers and mating frequency. This suggests that the estrogen receptor in the male is involved in regulating spermatogenesis, reproductive behavior, and epididymal function. Differential display is being used to identify genes with different levels of expression in the testis and epididymis of juvenile and adult ERKO and wild-type mice. However, changes in gene expression defining the mechanisms that require estrogen action in male reproduction have not yet been identified. Other studies focus on maturation of sperm in the epididymis. As sperm transit the epididymis, they acquire the ability to bind to the zona pellucida of the egg, to display a vigorous and coordinated pattern of flagellar motility, and to undergo capacitation and the acrosome reaction. These processes are being examined in ERKO mice to determine if they are estrogen-regulated and if modifications in these processes are a cause of infertility in ERKO males. Future studies will examine the role of the recently identified estrogen receptor beta in these processes.